Confocal Imaging of Cells Treated with mNPs

For confocal imaging analysis, we followed the protocol published before [17 (link)]. Briefly, 1 × 10⁵ cells were grown on glass coverslips with polylysine, incubated with mNPs for 24 h, and then washed twice with 1 mL of PBS, fixed for 20 min in 3.7% paraformaldehyde in PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were treated in a blocking buffer (3% BSA and 0.1% Triton X-100 in PBS) for 1h at rtand stained with phalloidin for 1 h. Hoechst33342 dye (Blue) (10 µg/mL) (Invitrogen, Waltham, MA, USA) was used for nuclei staining for 15 min incubation at rt. An anti-fade mounting medium (Biomeda, Foster City, CA, USA) was used for observing the labeled cells under a Bio-Rad MRC 1024 ES Confocal Laser Scanning Microscope (Bio-Rad, Hercules, CA, USA) equipped with a 15 mW Krypton/Argon laser source for fluorescence measurements (Figure 1). Cells were examined with a Nikon Plan Apo X60oil immersion objective using an excitation wavelength appropriate for Alexa 488 (495 nm). A series of optical sections (XY: 512 × 512 pixels) were then taken through the depth of the cells with a thickness of 1 µm at intervals of 0.8 µm (Z step). A single composite image was obtained by the superimposition of twenty optical sections for each sample [17 (link)].